1. Field of the Invention
The present invention relates to generation of novel thrombolytic molecules with enhanced protein transduction efficiency and processes therefor.
2. Description of the Related Art
Occlusion of blood vessels occurs due to the presence of blood clots (thrombi). Thrombin that is composed of fibrin and blood cells, may form in any part of the cardiovascular system including the veins, arteries, heart and microcirculation (Badimon at al., J. Clin. Invest. 1989, 84, 1134-1144). Thrombi progressively undergo structural changes; leucocytes that are attracted by chemotactic factors released from the aggregated platelets or proteolytic fragments of plasma proteins become incorporated into the thrombi. These aggregated platelets swell and disintegrate and are gradually replaced by fibrin. These clots often affect heart and lungs and may loosen and block smaller blood vessels.
Normal and timely blood flow through the damaged blood vessels requires the use of fibrinolytic agents. See, e.g., Chesbro et al., Circulation, 1987, 76, 142-154.
Streptokinase, staphylokinase, and other thrombolytic agents such as urokinase and tissue plasminogen activators are commonly used in the treatment of myocardial infarction, pulmonary, arterial or venous thromboembolism, surgical adhesions and other such instances when thrombi are formed. Thrombolytic agents act by converting endogenous plasminogen (a proenzyme) to plasmin (an active enzyme), which lyses the clot and could be used as thrombolytic agent in vivo. Plasminogen is a single chain glycoprotein, which in its native form has an amino terminal glutamic acid. It is converted into plasmin by the cleavage of Arg-Val (560-561) peptide bond. Robbins et al., Methods in Enzymology, 1976, 45, 257-273.
In the case of cerebral hemorrhage, smaller diseased arteries may rupture and bleed into the brain. Both of these events damage the brain and are collectively referred to as strokes (cerebrovascular accidents or CVAs). A cerebral stroke normally produces a sudden onset of symptoms. Depending on the artery affected, symptoms can include paralysis, speech difficulties, and difficulty in swallowing, visual and sensory disturbances.
Cerebral thrombosis can be treated by invasive procedures that can be highly traumatic and often have low therapeutic efficiency with substantial side effects. One of the alternative methods of treating stroke could involve employing one or more fibrinolytic agents such as streptokinase, acylated plasminogen-streptokinase complex, staphylokinase, urokinase, tissue plasminogen activator, and the like in cerebral tissue.
However there is a limitation in the uptake of such protein molecules into brain because of the blood-brain barrier that is impervious to most if not of all the proteins.
Blood brain barrier is the limiting factor in virtually all brain drug development programs since >98% of all small molecules and 100% of the large protein molecules do not cross the blood brain barrier. At the molecular level, the blood brain barrier consists of microvascular endothelial cells lining the brain microvessels together with closely associated astrocytic end feet processes. The microcapillary endothelium is characterized by the presence of tight junctions between the cerebral endothelial cells that form a diffusion barrier, which selectively excludes most blood-borne substances from the brain tissue.
The distribution of drug in brain requires a transvascular route and this approach requires the ability to undergo transport across the blood brain barrier. There has been success in transduction of protein molecules across the blood brain barrier by fusing or synthesizing proteins with protein transduction domain (PTD) to transport hydrophilic cargoes. Transduction peptides, in particular SynB (Rousselle et al., J. Pharmacol. Exp. Ther., 2001, 296, 124-131), Penetratin (Mazel et al., Anticancer Drugs, 2001, 12, 107-116; Rousselle et al., Mol. Pharmacol., 2000, 57, 679-686), and TAT peptides (Schwarze et al., Science, 1999, 285, 1569-1572; Cao et al., J. Neurosci. 2002, 22, 5423-5431; and Asoh et al., Proc. Natl Acad. Sci USA, 2002, 99, 17107-17112) markedly increase access to the brain. For example, targeting anti-apoptotic peptides (Cao et al., 2002; Asoh et al. 2002) into the brain has been used to provide protection against ischemic injury. A homeodomain-derived peptide to internalize C3-transferase (the small GTP-binding protein that antagonizes Rho) and reversed neuronal death in the spinal cord by 50% after injury (Mainguy et al., Nature Biotechnol., 2000, 18, 746-749). A cell penetrating peptide targeting eukaryotic initiation factor has been used to induce apoptosis in cancer cells (Herbert et al., Curr. Biol., 2000, 10, 793-796).
The use of cell penetrating peptides in the target molecules offers an unlimited scope for protein therapeutics. This invention is one such example of using this technique for designing novel thrombolytic molecules for dissolving thrombi in vasculature and in tissues including the cerebral tissues for indications of cerebral thrombosis.